Method of activation of oxazaphosphorines

ABSTRACT

The present invention provides a method of hydroxylating or oxidizing a compound of interest in a subject (e.g., a cytotoxic oxazaphosphorine prodrug), by administering the compound of interest to the subject; and concurrently administering the subject a metalloporphyrin in an amount effective to hydroxylate or oxidize the compound of interest in the subject.

RELATED APPLICATIONS

This application is a national phase application of PCT ApplicationPCT/US2007/023359, filed Nov. 6, 2007, and published in English on Jun.19, 2008, as International Publication No. WO 2008/073195, and whichclaims the benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication Ser. No. 60/864,816, filed Nov. 8, 2006.

GOVERNMENT FUNDING

The present invention was made with government support under grant nos.5-P 30-CA14236-29 from the NIH/NCI, BC024326 from the DOD, and CA16783from the NCI Public Health Services Department of Health and HumanServices. The US Government has certain rights to this invention.

FIELD OF THE INVENTION

This invention concerns methods and formulations useful for thetreatment of cancer in human and animal subjects in need thereof.

BACKGROUND OF THE INVENTION

Cyclophosphamide (CP), an oxazaphosphorine-type of cytotoxic pro-drug(including ifosfamide, mafosfamide, and trofosfamide), is the singlemost commonly utilized component in (i) conventional chemotherapy and(ii) high-dose chemotherapy/bone marrow transplant/stem-cell rescueregiments for cancer treatments [1-5]. The activation through thehydroxylation of CP by microsomal cytochrome P450 enzymes in liver,leads to 4-hydroxycyclophosphamide (4-OHCP) (FIG. 1), and ultimately tothe formation of cytotoxic phosphoramide mustard which alkylates DNA,thus preventing the proliferation of tumor cells [1-5]. There are,however, side reactions on this pathway leading to the enzymeinactivation and toxic byproducts causing systemic toxicity. Thus, inorder to increase the efficacy of the pro-drug at less toxic doses, itwould be beneficial to localize the production of cytotoxic metabolitesat the tumor site.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of hydroxylating oroxidizing a compound of interest in a subject (e.g., a cytotoxicoxazaphosphorine prodrug), comprising: administering the compound ofinterest to the subject; and concurrently administering the subject ametalloporphyrin in an amount effective to hydroxylate or oxidize thecompound of interest in the subject.

Stated otherwise, the present invention provides, in a method oftreating a subject in need thereof with a cytotoxic oxazaphosphorineprodrug, the improvement comprising concurrently administering thesubject a metalloporphyrin in an amount effective to enhance theefficacy of the oxazaphosphorine in the subject.

A further aspect of the invention is a pharmaceutical compositioncomprising, consisting of, or consisting essentially of: a cytotoxicoxazaphosphorine prodrug; a metalloporphyrin in an amount effective toenhance the efficacy of the oxazaphosphorine in a subject; and apharmaceutically acceptable carrier.

A still further aspect of the present invention is the use of an activeagent metalloporphyrin as described above for the preparation of amedicament for the treatment of a disorder as described above.

The present invention is explained in greater detail in the drawingsherein and the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Cytochrome P450 and Fe(III) porphyrin-catalyzed hydroxylation ofcyclophosphamide.

FIG. 2. The hydroxylation of 1 mM cyclophosphamide at 6 hours by 10 μMmetalloporphyrins and corresponding metal chlorides in the presence of 2mM ascorbate, in PBS at pH 7.4 (black bars), and pH 5.5 (gray bars) at37° C. under aerobic conditions (0.26 mM oxygen). Errors are ±2%.

The present invention is explained in greater detail in the followingnon-limiting Examples. The disclosures of all US Patent references citedherein are to be incorporated herein by reference in their entirety.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

“Cancer” as used herein includes any type of cancer, including but notlimited to lung, colon, colorectal, liver, breast, prostate, ovarian,brain, and skin cancers or tumors.

“Autoimmune disease” as used herein includes any type of autoimmunedisease, examples of which include but are not limited to arthritis,type 1 insulin-dependent diabetes mellitus, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, thromboticthrombocytopenic purpura, Sjogren's syndrome, encephalitis, uveitis,leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever,Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis,primary biliary cirrhosis, pemphigus, pemphigoid, necrotizingvasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, CNS inflammatory disorder, antigen-antibody complex mediateddiseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Gravesdisease, habitual spontaneous abortions, Reynard's syndrome,glomerulonephritis, dermatomyositis, chronic active hepatitis, celiacdisease, tissue specific autoimmunity, degenerative autoimmunity delayedhypersensitivities, autoimmune complications of AIDS, atrophicgastritis, ankylosing spondylitis and Addison's disease. See, e.g., U.S.Pat. No. 6,984,625.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the disease, etc.

The term “pharmaceutically acceptable” as used herein means that thecompound or composition is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the severity of the disease and necessity of thetreatment.

As used herein, the administration of two or more compounds“concurrently” means that the two compounds are administered closelyenough in time that the presence of one alters the biological effects ofthe other. The two compounds may be administered simultaneously orsequentially. Simultaneous administration may be carried out by mixingthe compounds prior to administration, or by administering the compoundsat the same point in time but at different anatomic sites and/or byusing different routes of administration.

The present invention is primarily concerned with the treatment of humansubjects, but the invention may also be carried out on animal subjects,particularly mammalian subjects such as mice, rats, dogs, cats,livestock and horses for veterinary purposes, and for drug screening anddrug development purposes.

1. Active Compounds.

The present invention employs oxazaphosphorine active compounds incombination with metallic porphyrin active compounds.

Oxazaphosphorines that may be used to carry out the present inventionare known. Examples include cyclophosphamides, which in turn includecyclophosphamide and derivatives and analogs thereof which are effectiveas a cancer chemotherapeutic agent through essentially the samemechanism or mode of action as cyclophosphamide. See, e.g., U.S. Pat.No. 6,268,138. Examples of such analogs and derivatives include but arenot limited to ifosfamide, maphosphamide, and trofosfamide. Additionalexamples are disclosed in U.S. Pat. Nos. 6,187,941; 5,190,929;4,770,870; and 4,716,242, the disclosures of which are incorporated byreference herein in their entirety.

Metalloporphyrins that may be used to carry out the present inventionare known. Numerous examples are available in the literature onporphyrins used in catalysis (see, e.g. References 12-40 herein) and theliterature on porphyrins used in photodynamic therapy. Examples includebut are not limited to those described in U.S. Pat. Nos. 7,087,214;7,067,653; 6,995,260; 6,953,570; 6,777,402; 6,573,258; 6,147,207;5,770,619; 5,424,305; 5,214,036; and 5,053,423. Where particular metalsare described as the coordinated metal in the porphyrins shown in theforegoing, those skilled in the art will appreciate that other metalscan be substituted in place thereof, with iron, manganese, cobalt,nickel, ruthenium, and copper currently preferred. Preferably themetalloporphyrin is selected to catalyze the hydroxylation of theconcurrently administered oxazaphosphorazine. Thus the term“metalloporphyrin” is herein intended to include transitionmetal(III)-substituted macrocyclic ligand complexes such as metal(III)complexes with substituted porphyrin and substituted porphyrin analoguessuch as are porphyrazine, texaphyrin, N-confused porphyrin and corrole.“Substituted macrocyclic ligand” is meant to include and not be limitedto substituted porphyrins, porphyrazines, texaphyrins, and corroles. Thesubstituted macrocyclic ligands of the presently disclosed subjectmatter include, for example, ortho-, meta- and para-tetrakisN-substituted porphyrins. The substituted macrocyclic ligands of thepresently disclosed subject matter include, for example, those compoundsdisclosed in WO 2005/077269 A1, U.S. Pat. Nos. 6,916,799 B2, 6,544,975B1, International Publication No. WO 00/075144 A3, InternationalPublication No. WO 2005/097123 A3 and Patent Application Publication No.US 2007/0072825 A1, all of which are herein incorporated by reference intheir entirety. Thus in some embodiments the metalloporphyrin isselected from the group consisting of a substituted porphyrin,porphyrazine, texaphyrin, and corrole. Particular examples include thosewherein the substituted porphyrin is an ortho-, meta- or para-tetrakisN-substituted porphyrin. More particular examples include those whereinthe ortho-, meta- or para-tetrakis N-substituted porphyrin is anortho-tetrakis N-alkylpyridinium porphyrin, wherein the alkyl has from1-8 CH₂ groups. More particular examples include those wherein theortho-tetrakis N-alkylpyridinium porphyrin is anortho-tetrakis(N-ethylpyridinium-2-yl])porphyrin or anortho-tetrakis(N-hexylpyridinium-2yl)porphyrin. Still more particularexamples include those wherein the ortho-, meta- or para-tetrakisN-substituted porphyrin is selected from the group consisting of atetrakis di(N,N′)-alkylimidazolium porphyrin, a tetrakisdi(N,N′)-alkylpyrazolium porphyrin, a tetrakisdi(N,N′)-alkylpyrimidinium porphyrin, a tetrakisdi(N,N′)-alkylpyrazinium porphyrin and a tetrakisdi(N,N′)-alkylpyridazinium porphyrin, wherein the alkyl has from 1-8 CH₂groups.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, commensurate with areasonable risk/benefit ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention. The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compound of the above formulae,for example, by hydrolysis in blood. A thorough discussion is providedin T. Higuchi and V. Stella, Prodrugs as Novel delivery Systems, Vol. 14of the A.C.S. Symposium Series and in Edward B. Roche, ed.,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are incorporated byreference herein. See also U.S. Pat. No. 6,680,299 Examples include aprodrug that is metabolized in vivo by a subject to an active drughaving an activity of active compounds as described herein, wherein theprodrug is an ester of an alcohol or carboxylic acid group, if such agroup is present in the compound; an acetal or ketal of an alcoholgroup, if such a group is present in the compound; an N-Mannich base oran imine of an amine group, if such a group is present in the compound;or a Schiff base, oxime, acetal, enol ester, oxazolidine, orthiazolidine of a carbonyl group, if such a group is present in thecompound, such as described in U.S. Pat. Nos. 6,680,324 and 6,680,322.

The active compounds disclosed herein can be prepared in the form oftheir pharmaceutically acceptable salts. Pharmaceutically acceptablesalts are salts that retain the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; (b) salts formed fromelemental anions such as chlorine, bromine, and iodine, and (c) saltsderived from bases, such as ammonium salts, alkali metal salts such asthose of sodium and potassium, alkaline earth metal salts such as thoseof calcium and magnesium, and salts with organic bases such asdicyclohexylamine and N-methyl-D-glucamine.

2. Pharmaceutical Formulations.

The active compounds described above may be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical formulationaccording to the invention, the active compound (including thephysiologically acceptable salts thereof) is typically admixed with,inter alia, an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the patient. Thecarrier may be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight ofthe active compound. One or more active compounds may be incorporated inthe formulations of the invention, which may be prepared by any of thewell known techniques of pharmacy comprising admixing the components,optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sublingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Preferred routes of parenteral administration include intrathecalinjection, including directly into the tumor, and intraventricularinjection into a ventricle of the brain.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound of theinvention, or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2Mactive ingredient.

Further, the present invention provides particulate formulations inwhich the active agent (or salt thereof) is encapsulated in orincorporated into a biodegradable particle, liposome or lipid vesicle,nanoparticle or the like. Techniques for forming such particulateformulations are known in the art. For example, when the compound orsalt thereof is an aqueous-soluble salt, using conventional formulationtechnology, the same may be incorporated into particles such as lipidvesicles. In such an instance, due to the water solubility of thecompound or salt, the compound or salt will be substantially entrainedwithin the center or core of a particle such as a liposome. The lipidlayer employed may be of any conventional composition and may eithercontain cholesterol or may be cholesterol-free. When the compound orsalt of interest is water-insoluble, again employing conventionalformation technology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome, orother particle space. In either instance, the particles which areproduced may be reduced in size, as through the use of standardsonication and homogenization techniques.

Of course, the particulate and/or liposomal formulations containing thecompounds disclosed herein or salts thereof, may be lyophilized toproduce a lyophilizate which may be reconstituted with apharmaceutically acceptable carrier, such as water, to regenerate aparticulate and/or liposomal suspension.

Other pharmaceutical compositions may be prepared from thewater-insoluble compounds disclosed herein, or salts thereof, such asaqueous base emulsions. In such an instance, the composition willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound or salt thereof.Particularly useful emulsifying agents include phosphatidyl cholines,and lecithin.

In addition to active compounds, the pharmaceutical compositions maycontain other additives, such as pH-adjusting additives. In particular,useful pH-adjusting agents include acids, such as hydrochloric acid,bases or buffers, such as sodium lactate, sodium acetate, sodiumphosphate, sodium citrate, sodium borate, or sodium gluconate. Further,the compositions may contain microbial preservatives. Useful microbialpreservatives include methylparaben, propylparaben, and benzyl alcohol.The microbial preservative is typically employed when the formulation isplaced in a vial designed for multidose use. Of course, as indicated,the pharmaceutical compositions of the present invention may belyophilized using techniques well known in the art.

3. Dosage and Routes of Administration.

As noted above, the present invention provides pharmaceuticalformulations comprising the active compounds, separately or incombination (including the pharmaceutically acceptable salts thereof),in pharmaceutically acceptable carriers for oral, rectal, topical,buccal, parenteral injection (e.g., intramuscular, intradermal,intravenous, intraarterial), and transdermal administration.

Routes of parenteral administration include intratumoral injection,intrathecal injection (including directly into a brain tumor), andintraventricular injection into a ventricle of the brain.

The therapeutically effective dosage of any specific compound, the useof which is in the scope of present invention, will vary somewhat fromcompound to compound, and patient to patient, and will depend upon thecondition of the patient and the route of delivery. As a generalproposition, a dosage from about 0.1 to about 50 mg/kg will havetherapeutic efficacy, with all weights being calculated based upon theweight of the active compound, including the cases where a salt isemployed. Toxicity concerns at the higher level may restrict intravenousdosages to a lower level such as up to about 10 mg/kg, with all weightsbeing calculated based upon the weight of the active base, including thecases where a salt is employed. A dosage from about 10 mg/kg to about 50mg/kg may be employed for oral administration. Typically, a dosage fromabout 0.5 mg/kg to 5 mg/kg may be employed for intramuscular injection.Preferred dosages are 1 μmol/kg to 50 μmol/kg, and more preferably 22μmol/kg and 33 μmol/kg of the compound for intravenous or oraladministration. The duration of the treatment is usually once per dayfor a period of two to three weeks or until the condition is essentiallycontrolled. Lower doses given less frequently can be usedprophylactically to prevent or reduce the incidence of recurrence of theinfection.

It also is conceivable that more than one administration of either anactive compound of the invention, or the other agent will be desired. Inthis regard, various combinations may be employed. By way ofillustration, where the active compound is “A” and the other agent is“B”, the following permutations based on 3 and 4 total administrationsare exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are likewise contemplated.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLE 1

This invention aims at developing suitable low-molecular weightsubstitute for the cytochrome P450 enzyme with the ultimate goal todeliver it directly to the tumor thus producing the active metabolite4-OHCP locally at high concentrations but at low systemic levels. Forthis purpose we utilize metalloporphyrins which exhibit highmetal-ligand stability and redox activity.

Metalloporphyrin (MP) constitutes the active site of numerous enzymes(hemoglobin, myoglobin, cytochromes, chlorophyll, guanylate cyclase,catalase, peroxidase, nitric oxide synthase, vitamin B₁₂, chlorophyll,etc), due to: (a) the ability of the metal center to bind oxygen (O₂)and other small molecules (CO, NO, H₂O), as well as many protein aminoacid residues (cysteine, methionine, histidine, etc) and (b) theelectron transfer (redox) properties of the metal, i.e. the ability toaccept and donate electrons thus acting as a catalyst [6-8].

In a laboratory setting, the core structure of the porphyrin ligand canbe modified to alter the chemical and redox properties of the metalcenter as well as the solubility and bulkiness of the whole molecule[6-9]. Synthetically prepared metalloporphyrins, highly stablelow-molecular-weight molecules capable of catalyzing electron-transferreactions, have been successfully utilized in industrial catalysis, asimaging contrast agents, in photodynamic therapy [10,11] and asbiological catalysts-enzyme mimics [12-20]. Both the Fe and Mnporphyrins have been studied as mimics of cytochrome P450 in oxidation,epoxidation, and hydroxylation in the presence of oxidants [21-31] andreductants/excess oxygen either in organic or biphasic systems [32-38].Mimicking cytochrome P450-catalyzed dehydration [39] and cytochrome P450reductase activity have been reported also [40].

To the best of our knowledge, the work presented here is the first studywhere low-molecular weight water-soluble metalloporphyrins have beenemployed as catalysts (mimics of liver cytochrome P450 enzymes) in ananti-cancer drug activation reaction, in an all-aqueous biologicallyrelevant medium without additional oxidant and with cellular reductantascorbate as a source of electrons (instead of NADPH in the case ofcytochrome P450), under concentrations similar to those found in vivo[41].

EXPERIMENTAL

Abbreviations

The following abbreviations are used herein: MP, metalloporphyrin;Fe^(III)T(2,6-F₂-3-SO₃—P)P³⁻, Fe(III) mesotetrakis(2,6-difluoro-3-sulfonatophenyl)porphyrin;Fe^(III)T(2,6-Cl₂-3-SO₃—P)P³⁻, Fe(III) mesotetrakis(2,6-dichloro-3-sulfonatophenyl)porphyrin; Fe^(III)TBAP³⁻, alsoabbreviated as Fe^(III)TCPP³⁻, Fe(III) mesotetrakis(4-carboxylatophenyl)porphyrin; Fe^(III)PP-IX⁻, Fe(III)protoporphyrin IX; Fe^(III)TM-2-PyP⁵⁺, Fe(III)tetrakis(N-methylpyridinium-2-yl)porphyrin; Mn^(III)TM-2-PyP⁵⁺, Mn(III)tetrakis(N-methylpyridinium-2-yl)porphyrin; Mn^(III)TM-3-PyP⁵⁺, Mn(III)tetrakis(N-methylpyridinium-3-yl)porphyrin; CP, cyclophosphamide;4-OHCP, 4-hydroxycyclophosphamide; NHE, normal hydrogen electrode; PBS,phosphate-buffered saline.

Materials. We employed two electron-deficient anionic Fe(III) mesotetrakis(2,6-difluoro-3-sulfonatophenyl)porphyrin(Fe^(III)T(2,6-F₂-3-SO₃—P)P³⁻), and Fe(III) mesotetrakis(2,6-dichloro-3-sulfonatophenyl)porphyrin(Fe^(III)T(2,6-Cl₂-3-SO₃—P)P³⁻), and two electron-rich anionic Fe(III)meso tetrakis(4-carboxyphenyl)porphyrin (Fe^(III)TBAP³⁻, alsoabbreviated as Fe^(III)TCPP³⁻) and Fe(III) protoporphyrin IX(Fe^(III)PP-IX⁻) (Scheme I). All porphyrins were obtained fromMidCentury Chemicals, Chicago, Ill. (Scheme I). Also, theelectron-deficient cationic Fe(III) mesotetrakis(N-methylpyridinium-2-yl)porphyrin), Fe^(III)TM-2-PyP⁵⁺, and itsMn(III) isomeric analogues, Mn^(III)TM-2-PyP⁵⁺ and Mn^(III)TM-3-PyP⁵⁺,prepared as previously described, were studied [42,42]. Although ofextremely high metal-ligand stability when its metal center is in the +3oxidation state, a metalloporphyrin may lose metal if metal center isreduced to the +2 state in reducing cellular environment [42,43]. Tostudy the possible effects of free metal on the hydroxylation ofcyclophosphamide, the metal salts FeCl₃ (FeCl₃×6H₂O, Mallinckrodt) andMnCl₂ (MnCl₂×4H₂O, J. T. Baker) were used as controls. Cyclophosphamide,D-mannitol, and sodium L-ascorbate were from Sigma, PBS was from Gibcoand H₂O₂ (30% solution) was from Mallinckrodt. Theo-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride was obtainedfrom Lancaster Synthesis.

Methods

The electrochemistry was done as previously described [42,43] on CHInstruments 600 Voltammetric Analyzer. The metal-centered redoxpotentials of aquametalloporhyrins were measured in 0.1 M NaCl and 0.01M HCl. The anionic porphyrins, FeTBAP³⁻ and FePP-IX⁻ precipitate underacidic conditions when carboxylates are protonated [42]. Thus, theirvoltammograms were obtained in 0.1 M NaCl, 0.1 M 1-methylimidazole, 0.05M phosphate buffer, pH 7.8. Scan rates were 0.1 V/s. The potentials werestandardized against MnTE-2-PyP⁵⁺ and are given in Table 1, in mV versusnormal hydrogen electrode, NHE [42].

Hydroxylation reaction was followed in phosphate-buffered saline (PBS)at pH 5.5 and 7.4 and at 37° C. with 10 μM Fe and Mn porphyrins, 2 mMascorbate and 1 mM cyclophosphamide, under aerobic conditions (0.21 mMO₂). The reaction was stopped by dilution with organic solvents and thehydroxylation product 4-OHCP was trapped overnight by a hydroxylamineforming a stable aldophosphamide oxime derivative as reported [4]. Theoxime was subsequently measured by GC/MS as previously described [4].

TABLE 1 The metal-centered redox potential of aquametalloporhyrins aredone in 0.1 M NaCl and 0.01 M HCl, and of FeTBAP³⁻ and FePP-IX⁻ weredone in 0.1 M NaCl, 0.1 M 1-methylimidazole, 0.05 M phosphate buffer, pH7.8. Scan rates were 0.1 V/s. Errors are ±3 mV. Charges were omittedwhen respective redox couples were indicated. M is either iron ormanganese. E_(1/2), mV vs NHE (H₂O)M^(III)P/ (OH)M^(III)P/(1-MeIm)₂M^(III)P/ Metalloporphyrin (H₂O)M^(II)P (H₂O)M^(II)P(1-MeIm)₂M^(II)P FeT(2,6-F₂-3-SO₃P)P³⁻ +124   +74^(a) +114^(a)FeT(2,6-Cl₂-3-SO₃P)P³⁻ +144   +94^(b) +134^(b) FeTM-2-PyP^(5+a) +355^(b)+212^(b) +432^(b) FeTBAP³⁻  +22 FePPIX⁻ 112 MnTM-2-PyP^(5+a) +220^(b)+128^(b) MnTM-3-PyP^(5+a)  +52^(b)  +42^(c) Fe^(III)/Fe^(II b) +771^(d)Mn^(III)/Mn^(II b) +1541^(d)  ^(a)data estimated based on the existingpotentials of difluoro- and dichloro analogues [42] and those obtainedherein. The di-ortho-substituted porphyrins are fairly insensitive toaxial ligation due to the steric hindrance when compared to themono-ortho porphyrins. ^(b)ref 42, data obtained at pH 7.8, 0.05 Mphosphate buffer, 0.1M NaCl, 0.5 mM porphyrin. ^(c)ref 62, data obtainedat pH 11 where only 33% of the (OH)MnTM-3-PyP⁴⁺ exists; pK_(a) of theaxial water is 11.5^([17])). ^(d)ref 63.

Generally, the synthetic antioxidants are prone to undergo oxidativedegradation, which limits their catalytic efficacy. That in turns makesa design of robust catalyst quite a challenge. The stability ofmetalloporphyrins with respect to oxidative degradation was followed onShimadzu UV-2501PC spectrophotometer under aerobic conditions similar tothose previously reported [43,49]: 10 μM porphyrins, 2 mM ascorbic acid,0.26 mM O₂, at 25° C. and in PBS, at pH 7.4 where both cationic andanionic porphyrins were soluble [42] and in the presence and absence of1 mM cyclophosphamide. In addition, the time-dependent spectra andtime-dependent change in absorbance at 417.5 nm ((H₂O)Fe^(II)TM-2-PyP⁴⁺)were followed with 9 μM (OH)Fe^(III)TM-2-PyP⁵⁺, 2 mM ascorbic acid,+/−50 μM H₂O₂, +/−50 mM mannitol (scavenger of OH radical), in PBS, pH7.4, under aerobic conditions (0.26 mM O₂) at 25° C. (data not shown).We have chosen FeTM-2-PyP⁵⁺ based on the study of Almarsson and Bruice[50] which showed that with multianionic and multicationic porphyrins,the charge surrounding iron metal center seems not to greatly influencethe reaction of iron porphyrins with alkylhydroperoxides.

All experiments were performed in triplicate.

Results and Discussion. A catalytic acceleration of the hydroxylationoccurred at 37° C. after the addition of 10 μM Fe(III) and Mn(III)porphyrins into the incubation mixture containing 1 mM cyclophosphamide,2 mM ascorbate and phosphate-buffered saline PBS (pH 7.4) (FIG. 2).Cyclophosphamide alone, without metalloporphyrin or ascorbate present,did not produce 4-OHCP. Also, metalloporphyrins alone did not cause anyCP hydroxylation. In the absence of metalloporphyrin, ascorbic acid,itself, underwent slow reaction with oxygen [44], leading to very lowyields (FIG. 2). The similar level of hydroxylation was observed withMnCl₂ in the presence of ascorbic acid and may thus be ascribed to theeffect of ascorbic acid alone. FeCl₃ was only slightly better than theascorbic acid alone in hydroxylating cyclophosphamide, which suggests aweak catalytic action of free iron.

A pH effect was observed with difluoro-, dichloroporphyrin andFeTM-2-PyP⁵⁺; the yield of 4-OHCP increased up to 3-fold in a pH 5.5medium (FIG. 2) where 10 μM FeT-2,6-F₂-3-SO₃—P³⁻ hydroxylated ˜5-foldmore of cyclophosphamide. The pK_(a) values for the axially ligatedwater vary between 5.5 for the electron-deficient and 7.0 for theelectron-rich Fe(III) porphyrins [43]. At lower pH, the aquairon(III)species predominates, and with higher E_(1/2) (Table 1) it is morereadily reducible than the monohydroxoiron(III) species [42].Consequently, a certain level of favorable selectivity is expected inthe production of 4-OHCP in tumor, whose extracellular pH was reportedto be as low as 5.2, when compared to normal tissue (pH ˜7.3) [45-48].Moreover, near the surface of macrophages, which may comprise up to 50%of the tumor mass, the pH may be as low as 3.6 [45-48]. At pH 5.5 where˜50% of carboxylate groups of FePP-IX⁻ and FeTBAP³⁻ (pK_(a) are ˜5) areprotonated, both porphyrins precipitate out of the solutions. Thus no pHeffect has been assessed with these two porphyrins. Due to the weakaxial interactions that dominate the chemistry of Mn porphyrins [42 andrefs therein], they exist in the form of aquamanganese(III) porphyrinspecies in a wide range of pH=0-9 [42] and are thus insensitive to a pHchange from 7.4 to 5.5 (FIG. 2).

We have previously observed that aerobically, in the presence of excessascorbic acid over oxygen, Mn porphyrins are reduced at the metal center(followed by a corresponding change in uv/vis spectra) and then undergofurther uv/vis spectral change [42,49] which is qualitatively identicalto the change observed upon their exposure to H₂O_(2 [)42].Time-dependent spectra and kinetic traces (data not shown) observed inthis work with Fe^(III)TM-2-PyP⁵⁺/ascorbate system (PBS, pH 7.4) wereessentially the same whether H₂O₂ or mannitol (an OH scavenger) wereadded or not. The (OH)Fe^(III)TM-2-PyP⁴⁺ species, with Soret band at 408nm, was reduced by ascorbic acid to (H₂O)Fe^(III)TM-2-PyP⁴⁺ which hadSoret band at 417.5 nm. The 417.5 nm band did not shift upon theaddition of either mannitol or H₂O₂. The absorbance at 417.5 nmdecreased slowly (indicating porphyrin degradation), and at nearlyidentical rates whether mannitol or H₂O₂ were present in the system(data not shown). That suggests that H₂O₂, whether added to the systemor produced endogenously by the reduction of Fe-bound oxygen, is a keyspecies involved in porphyrin degradation which is in competition withhydroxylation. The lack of effect of mannitol indicates thatO═Fe^(IV)P.⁺ is hydroxylating cyclophosphamide rather than freelydiffusing .OH. Our data are best accommodated by the studies ofAlmarsson and Bruice [50], Murata et al [51] and Panicucci and Bruice[52]. The initial oxygen binding to the ascorbate-reduced Fe^(II)porphyrin center generates O₂ ⁻—Fe^(III)P, which is further reduced toFe(III) hydroperoxo species similar to cytochrome P450 [12]. Fe(III)hydroperoxo species undergoes O—O homolysis, leading to the solventcaged intermediates [H₃O⁺, O═Fe^(IV)P, OH], followed by 1e⁻ oxidation ofO═Fe^(IV)P by OH to yield O═Fe^(IV)P⁺, the latter species beingthermodynamically favored in aqueous solution [52].

The destruction (“bleaching”) of the porphyrin is a serious problemencountered in all studies of metalloporphyrin-based catalysis. Even theoxidative damage of hemoglobin has been recently reported [60]. Weobserved bleaching in the cases of all metalloporphyrins studied. Underthe same experimental conditions as in catalysis experiments (pH 7.4),the degradation of metalloporphyrins was followedspectrophotometrically. The most potent catalysts, difluoro- anddichloroporphyrins were the least stable. In the presence ofcyclophosphamide, after 6 hours the remaining fractions were: 19% ofFeT(2,6-F₂-3-SO₃P)P³⁻, 23% of FeT(2,6-Cl₂-3-SO₃P)P³⁻, 24% of FeTBAP³⁻,41% of FeTM-2-PyP⁵⁺, 71% of FePP-IX⁻, 91% of MnTM-2-PyP⁵⁺ and 94% ofMnTM-3-PyP⁵⁺. All porphyrins were more stable in the presence than inthe absence [42,43,49] of the substrate, cyclophosphamide. Thus,modulating the susceptibility of the metalloporphyrins to oxidation isimportant to maximize their catalytic potency/lifetime ratio.

Although difluoro- and dichloroporphyrins have similar E_(1/2) values(Table 1), the difluoroporphyrin proved to be the more effectivecatalyst in our studies. Steric hindrance in the case of theFeT(2,6-Cl₂-3-SO₃P)P³⁻, due to the bulky chlorine atoms in orthopositions, may account for its lower efficacy relative to the difluoroanalogue (FIG. 2). FeTM-2-PyP⁵⁺ is less potent than difluoro- anddichloroporphyrin. The more positive E_(1/2) of FeTM-2-PyP⁵⁺ (Table 1)stabilizes it in the Fe +2 oxidation state, which does not favorreoxidation by oxygen, thus lowering its hydroxylation power. However,its stability to bleaching partly compensates for its lower efficacy.Along this line, FePP-IX⁻ is stabilized in the Fe +3 oxidation state,and is consequently very resistant to bleaching. FeTBAP³⁻ with a morepositive E_(1/2) than FePP-IX⁻ is a more effective catalyst but again itis more prone to destruction. Mn porphyrins, MnTM-2-PyP⁵⁺ andMnTM-3-PyP⁵⁺ do not favor axial binding of the oxygen upon reduction andare thus inferior as catalysts to their Fe(III) analogue FeTM-2-PyP⁵⁺[42]. The meta isomer, MnTM-3-PyP⁵⁺ (E_(1/2)=+52 mV vs NHE) is moresusceptible to the reoxidation than ortho isomer MnTM-2-PyP⁵⁺(E_(1/2)=+228 mV vs NHE) which explains its higher catalytic potency(FIG. 2). It should be noted that the redox potential for theO═Mn^(IV)P/Mn^(III)P redox couple is insensitive to theelectron-deficiency/richness of the Mn porphyrin and consequently doesnot correlate with the observed differences in catalytic potency [42 andrefs therein].

The yield of 4-OHCP obtained with FeT(2,6-F₂-3-SO₃P)P³⁻/ascorbateapproximates well the yield obtained with cyt P450 enzymes in vivo.Thus, with 1 mM CP the 46.11 μM 4-OHCP was produced at pH 5.5, i.e. theyield was 4.6%. At pH 7.4 the 15.62 μM 4-OHCP was formed, i.e. the yieldwas 1.6% (FIG. 2). The cyclophosphamide concentration (1 mM) in oursystem equals those administered to patients (1.1 g) [5]. Based on thereported plasma data, after intravenous administration of 1.1 g of CP[5], around 2.4% of CP was hydroxylated to 4-OHCP (0.69 μg/mL) at thepeak CP levels (27.58 μg/mL μM).

In conclusion, the usefulness of a metalloporphyrin as cytochrome P450mimic would depend upon the combined effects of: (a) the optimalmetal-centered redox potential for the M^(III)P/M^(II)P redox couple,i.e. reducibility of M^(III)P; (b) the oxygen binding affinity of theM^(III)P species which is influenced by steric and electrostaticfactors; and (c) the susceptibility of the catalyst towards oxidativedegradation.

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The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A pharmaceutical composition comprising: a cytotoxic oxazaphosphorineselected from the group consisting of cyclophosphamide, ifosfamide,mafosfamide, trofosfamide, and pharmaceutically acceptable saltsthereof; a metalloporphyrin separate from said cytotoxicoxazaphosphorine, said metalloporphyrin included in an amount effectiveto enhance the efficacy of said oxazaphosphorine in a subject, whereinsaid metalloporphyrin comprises a transition metal(III)-substitutedmacrocyclic ligand complex containing a coordinated metal selected fromthe group consisting of iron, manganese, cobalt, nickel, ruthenium, andcopper; and a pharmaceutically acceptable carrier.
 2. The composition ofclaim 1, wherein said oxazaphosphorine comprises cyclophosphamide; andsaid metalloporphyrin comprises Fe^(III)T(2,6-F₂-3-SO₃—P)P³/Fe(III) mesotetrakis(2,6-difluoro-3-sulfonatophenyl)porphyrin.
 3. The composition ofclaim 1, wherein said cytotoxic prodrug and said metalloprophyrin arecarried by lipid particles.
 4. The composition of claim 1 in the form ofa tablet or capsule.